Programmed cell death or apoptosis is a physiological process essential for normal development and tissue homeostasis. Cell death mechanisms are protective measures for organisms which ensure the removal of unnecessary, damaged or potentially dangerous cells. However, any deregulation or inappropriate induction of this process leads to the loss of healthy cells, causing diseases. In particular, cell death in post-mitotic tissues such as the brain and heart in adult organisms results in functional compromise, as is the case in Alzheimer's disease, Parkinson's disease and stroke. Cell death induced by oxidative stress has been shown to be involved in the development of these pathologies. Although the exact mechanism of cell death induced by oxidative stress is still not known, mitochondria have been shown to play a central role in this process. Mitochondrial events such as opening of the permeability transition pores, mitochondrial membrane potential collapse and release of pro-apoptotic factors such as cytochrome c and/or apoptosis-inducing factors trigger the cascade of events leading to execution of apoptosis.
Bax is a 24 kDa protein of the Bcl-2 family with pro-apoptotic function. It normally resides in cytosol and translocates to mitochondria upon induction of apoptosis and it plays a key role in destabilizing mitochondria. Translocation of Bax to mitochondria followed by a conformational change (mitochondrial permeabilization) in association with Bid leads to the release of cytochrome c, apoptosis-inducing factor and caspase-9, a cysteine protease, which start the execution phase of apoptosis. Bax has been implicated in neuronal cell death during development and ischemia.
Caspase-3 is normally present in a dormant form. Once activated, it plays a role in the disintegration of various key proteins in the cell, including the activation of an endonuclease which fragments cell DNA.
Intrabodies to apoptotic proteins with inhibitory action would be useful in the treatment of neurodegenerative disorders, in addition to being valuable tools for studying apoptosis. The efficacy of intrabodies critically depends on their stability. In the reducing environment of the cytoplasm, intrabodies cannot form their stabilizing disulfide linkage(s), so only those which are of sufficient stability can tolerate the absence of the disulfide linkage and be expressed in functional form. Traditionally, single chain Fvs (single chain Fvs, or “scFvs” consist of an antibody heavy chain variable domain, VH, and a light chain variable domain, VL, joined together by a linker) have been used as intrabodies (Kontermann, R. E., 2004). More recently, the feasibility of three types of single-domain antibodies (sdAbs), VLs, VHs and VHHs (VHs derived from camelid heavy chain antibodies (Hamers-Casterman C. et al., 1993), as intrabodies has also been demonstrated. While offering a comparable affinity, sdAbs have higher stability, solubility and expression level than scFvs and thus, are more efficacious as intrabodies (Tanaka, T. et al., 2003; Aires da Silva, F. et al., 2004; Colby, D. W. et al., 2004) Intrabodies can be derived from monoclonal antibodies or antibody display libraries, e.g., antibody phage display libraries (Rondon, I. J. et al., 1997; Miller, T. W. et al., 2005).